Elucidating mechanisms that regulate erythroid cell maturation is critical for devising targeted therapies for red cell disorders including thalassemias, polycythemia vera and anemias, and for developing strategies to generate red blood cells for clinical transfusion. The PI3-kinase-AKT pathway promotes and suppresses erythroid maturation. The inhibitory mechanism involves AKT-mediated phosphorylation of FoxO3, which leads to its sequestration in the cytoplasm. FoxO3 is upregulated/activated during erythroid maturation and by oxidative stress and controls the erythroid cell cycle and maturation, red blood cell lifespan, and protects against oxidative stress. We developed evidence that the serine/threonine kinase mTOR engages in crosstalk with FoxO3 to control erythroid maturation. Mechanisms underlying FoxO3- mTOR crosstalk in any context are poorly understood. FoxO3 and mTOR were known to have critical independent functions to control autophagy in non-erythroid cells. Autophagy mediates the consumption of damaged cellular components and differentiation-associated cellular remodeling, including mitochondria disposal as a key step in erythrocyte development. We discovered that FoxO3 facilitates GATA-1-instigated autophagy gene activation and accumulation of the autophagosome during erythroid maturation. GATA-1 also activates autophagy by directly inducing FoxO3 expression. We hypothesize that GATA-1-FoxO3 cooperativity controls autophagy and is modulated by mTOR signaling, which would constitute a new paradigm with considerable potential for therapeutic modulation of erythropoiesis and understanding erythroid pathophysiologies. The Bresnick and Ghaffari groups will collectively test this hypothesis and elucidate mechanisms. Aim 1 - To elucidate mechanisms underlying FoxO3 regulation of terminal erythroid maturation. We will test the hypothesis that FoxO3 has a critical function to promote autophagy in late erythroid maturation. We will determine whether FoxO3 stimulates autophagy in steady-state and stress erythropoiesis contexts, if oxidative stress influences erythroid maturation by controlling autophagy, and if mTOR-FoxO3 crosstalk regulates autophagy. As inhibition of mTOR signaling improves anemia in a b-thalassemia model, we will address the potential function of autophagy in b-thalassemia. Aim 2 - To discriminate among models to explain how GATA-1 and FoxO3 cooperatively control autophagy and erythroid maturation. We hypothesize that GATA-1-FoxO3 cooperativity represents a physiological mechanism to control erythroid maturation. We will distinguish between models to understand the cooperativity, which conforms to a type I coherent feed-forward loop, and will determine how it contributes to establishment/maintenance of the erythroid genetic network. Given the importance of GATA and FoxO factors for regulating diverse processes, the studies will yield broad mechanistic and biological insights.